Defects in replication fidelity of simple repeated sequences reveal a new mutator mechanism for oncogenesis.
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Colorectal Neoplasms↗
Biomedical subjects
M Perucho
Publications and source records attributed to M Perucho.
Genetic heterogeneity of the c-K-ras locus in colorectal adenomas but not in adenocarcinomas.
BACKGROUND: Previous molecular genetics studies of colorectal cancer have identified multiple mutations in the c-K-ras gene (also known as KRAS2) in all phases of its development. Because of technical difficulty, prior studies rarely focused attention on the detailed distribution of c-K-ras mutations in multiple regions of the same primary tumor specimen. However, with recent development of the selective UV radiation fractionation method, characterization of c-K-ras mutations in multiple regions of the same primary tumor specimen can be performed. PURPOSE: Our purpose was to describe how c-K-ras mutations were distributed among cells obtained from multiple regions of the same primary tumor in an attempt to describe differences between early and late colorectal carcinogenesis. METHODS: Formalin-fixed, paraffin-embedded tissue blocks were obtained. Seven adenocarcinomas and seven adenomas were selected for the presence of mutant c-K-ras genes and histologic transitions between normal and neoplastic tissue. Tissue sections were prepared for analysis by the selective UV radiation fractionation method by placing thin, fixed tissue sections on a plastic slide with no coverslip. Under the microscope, small ink dots from a felt-tip pen were manually placed directly on relatively pure cell subpopulations. The slides were placed with the tissue side exposed to a UV transilluminator for 2-4 hours to inactivate the DNA present in the unprotected ("undotted") cells. Individual dots were cut out of the plastic slide into 2 x 2-mm squares and placed into microfuge tubes. The DNA was extracted and supernatant used for polymerase chain reaction (PCR) analysis. Mutations at c-K-ras codons 12 and 13 were detected. RESULTS: The selective UV radiation fractionation method and PCR analysis revealed that c-K-ras mutations never extended into normal mucosa and were present in all neoplastic cells regardless of phenotypes in all seven adenocarcinomas and three of the seven adenomas. Further examination of two carcinomas for p53 (also known as TP53) mutations or loss of heterozygosity demonstrated that these additional mutations were also present in all tumor cells, suggesting that a single transformed clone was responsible for the majority of growth. However, in four other adenomas, tumor heterogeneity was demonstrated, since c-K-ras mutations were detected only in discrete portions. CONCLUSIONS: Adenoma formation may include a stage in which multiple and genetically distinct neoplastic clones are present, while most carcinomas appear to have a homogeneous composition that may result from the successful progression of one of these clones.
Adenocarcinoma↗
Ubiquitous somatic mutations in simple repeated sequences reveal a new mechanism for colonic carcinogenesis.
Spontaneous errors in DNA replication have been suggested to play a significant role in neoplastic transformation and to explain the chromosomal alterations seen in cancer cells. A defective replication factor could increase the mutation rate in clonal variants arising during tumour progression, but despite intensive efforts, increases in tumour cell mutation rates have not been unambiguously shown. Here we use an unbiased genomic fingerprinting technique to show that 12 per cent of colorectal carcinomas carry somatic deletions in poly(dA.dT) sequences and other simple repeats. We estimate that cells from these tumours can carry more than 100,000 such mutations. Only tumours with affected poly(dA.dT) sequences carry mutations in the other simple repeats examined, and such mutations can be found in all neoplastic regions of multiple tumours from the same patient, including adenomas. Tumours with these mutations show distinctive genotypic and phenotypic features. We conclude that these mutations reflect a previously undescribed form of carcinogenesis in the colon (predisposition to which may be inherited) mediated by a mutation in a DNA replication factor resulting in reduced fidelity for replication or repair (a 'mutator mutation').
Adult↗
Isolation and characterization of allelic losses and gains in colorectal tumors by arbitrarily primed polymerase chain reaction.
The arbitrarily primed polymerase chain reaction (AP-PCR) [Welsh, J. & McClelland, M. (1990) Nucleic Acids Res. 18, 7213-7218] has been used to detect somatic genetic alterations in tumors of the colon and rectum. DNA fingerprints generated by single arbitrary primers were compared between normal and tumor tissue of the same individuals. AP-PCR bands showing decreased and increased intensities in tumor tissue DNA, relative to normal, have been cloned after reamplification with the same arbitrary primer. Standard restriction fragment length polymorphism and Southern blot analyses show that these DNA sequences have undergone allelic losses and gains, respectively, in the tumor cell genome. The deleted sequences have been assigned to the short arm of chromosome 17 by PCR of somatic hamster/human cell hybrids and linkage analysis. These results show the ability of the AP-PCR to detect and isolate, in a single step, DNA sequences representing two of the genetic alterations that underlie the aneuploidy of cancer cells: losses of heterozygosity and chromosomal gains. Altogether, they also show the quantitative nature of the amplification levels obtained in vitro by AP-PCR, which thus provides the basis for an alternative molecular approach to cancer cytogenetics.
Adenocarcinoma↗
Differential expression of the normal and mutated K-ras alleles in chemically induced thymic lymphomas.
The presence of point mutations in the K-ras gene was examined in murine thymic lymphomas induced by a single dose of N-methylnitrosourea by the RNase A mismatch cleavage method and by allelic-specific oligonucleotide hybridization of in vitro amplified DNA by polymerase chain reaction. The results show that the frequency of mutations is lower than that of tumors induced by multiple N-methylnitrosourea treatments. Four mutations identified were the aspartic acid at codon 12, a G:C to A:T transition in its second position. A G:C to T:A transversion in codon 146 was also found in one thymic lymphoma, changing the amino acid alanine to serine. The use of the RNase A assay allowed an estimation of the relative expression levels of both normal and mutant K-ras alleles. The results show that in approximately one half of the tumors the mutant allele is predominantly expressed, suggesting that the normal allele has been lost or that the mutant allele has been amplified relative to the normal. Altogether, these findings are consistent with ras mutations occurring in some instances during tumor development and with a ras effect being not strictly dominant but favoring selection for increasing levels of expression from the oncogenic allele.
Animals↗
Characterization of genetic variation and 3'-azido-3'-deoxythymidine- resistance mutations of human immunodeficiency virus by the RNase A mismatch cleavage method.
The RNase A mismatch cleavage method has been applied to the characterization of natural genetic variation of human immunodeficiency virus (HIV) from different geographical areas. The approach provides a rapid and simple assay for the analysis of differences in closely related viral isolates and allows the establishment of phylogenetic relationships between epidemiologically distinct viruses. Our results show a broad clustering of circulating viruses according to their geographical distribution. We also have analyzed the temporal appearance of mutations associated with the acquisition of resistance to 3'-azido-3'-deoxythymidine (AZT). The results show that mutations in codon 215 of the viral reverse transcriptase can be detected readily by this method in HIV isolates and also directly in peripheral blood from HIV-infected individuals after in vitro amplification of viral sequences with the polymerase chain reaction. The specific recurrence of identical double-nucleotide substitutions in epidemiologically and geographically distant viruses suggests that the restricted amino acid substitutions at this position selected by drug exposure are a critical, rate-limiting step in the acquisition of drug resistance.
Acquired Immunodeficiency Syndrome↗
Frequency and spectrum of mutations at codons 12 and 13 of the c-K-ras gene in human tumors.
The frequency of point mutations at codons 12 and 13 of the c-K-ras gene has been determined in a panel of more than 400 human tumors. Mutant c-K-ras genes were detected in about 75% of adenocarcinomas of the pancreas (n = 84); 40% of adenomas (n = 72) and carcinomas (n = 244) of the colon end rectum; 30% of carcinomas of the bile duct (n = 19); 25% of carcinomas of the lung (n = 92), and in lower frequency in other carcinomas, including liver, stomach, and kidney. No mutations were found in carcinomas of the breast, prostate, esophagus, and gall bladder, among others. Comparative analysis of the spectrum of mutations show that while G to A transitions were the most frequent mutations in pancreatic and colo-rectal tumors, G to T transversions were more prevalent in lung carcinomas. The aspartic acid mutation at codon 13 (GGC----GAC) was relatively frequent in colo-rectal tumors but rare in pancreatic and lung carcinomas. The differences in the mutation spectrum of the c-K-ras gene in cancers of the gastrointestinal and respiratory tracts are suggestive of differential exposure to genotoxic agents.
Base Sequence↗
Detection of c-K-ras mutations in fine needle aspirates from human pancreatic adenocarcinomas.
Formalin-fixed paraffin-embedded tissue specimens obtained by fine needle aspiration of pancreatic masses from 47 patients were examined retrospectively for cytology and the presence of mutant c-K-ras oncogenes. Point mutations of c-K-ras in codon 12 were detected by RNA-DNA RNAse A mismatch cleavage after in vitro DNA amplification of the cellular c-K-ras sequences by the polymerase chain reaction. Of the 36 patients with pancreatic adenocarcinoma, mutant c-K-ras oncogenes were detected in 18 of 25 (72%) with malignant cytologies, 2 of 8 (25%) with atypical cytologies, and 0 of 3 with benign aspiration cytologies. The remaining 11 patients without pancreatic adenocarcinomas did not have mutant c-K-ras genes detectable by the assay. The diagnosis of pancreatic adenocarcinoma was based upon clinical follow-up. The presence of mutant c-K-ras oncogenes did not significantly affect survival in the patients studied. Mutant c-K-ras genes were found at the time of initial clinical presentation in the majority of pancreatic adenocarcinomas, suggesting an important role of the mutation in oncogenesis. In conjunction with cytology, our approach represents an application for cancer diagnosis at the molecular genetic level.
Adenocarcinoma↗
Mutational activation of the c-K-ras gene in human pancreatic carcinoma.
We have reported the presence of c-K-ras oncogenes activated by single point mutations at codon 12 in a vast majority of human pancreatic carcinomas. Formalin-fixed, paraffin-embedded specimens from surgical resections, autopsies and biopsies were used as well as snap frozen surgical specimens. The high oncogene incidence has been confirmed in other studies and indicate that somatic mutational activation of the c-K-ras gene is an important event in the development, maintenance or progression of cancer of the exocrine pancreas. While the role that these point mutations play in any or all of these processes remains to be determined, their presence is useful clinically for the diagnosis of pancreatic carcinoma at the molecular genetic level. The detection of mutated c-K-ras oncogenes in fine needle aspirates of pancreatic masses, that by cytomorphology may be suspicious but not diagnostic of malignant disease, increases the sensitivity of the diagnosis for this cancer. The identification of codon 12 mutations in the c-K-ras gene in pancreatic adenocarcinomas has been possible by advances in recombinant DNA techniques, especially by the development of in vitro gene amplification by the polymerase chain reaction (PCR). The possibility of analysing formalin-fixed, paraffin-embedded tissue for the presence of genetic alterations as small as single point mutations by PCR in concert with other mutation detection techniques, should facilitate the molecular genetic analysis of pancreatic carcinoma. Retrospective studies using stored specimens are now feasible with the technology described and should yield important information on the molecular epidemiology and aetiology of this and other diseases.
Adenocarcinoma↗
Most human carcinomas of the exocrine pancreas contain mutant c-K-ras genes.
Using in vitro gene amplification by the polymerase chain reaction (PCR) and mutation detection by the RNAase A mismatch cleavage method, we have examined c-K-ras genes in human pancreatic carcinomas. We used frozen tumor specimens and single 5 micron sections from formalin-fixed, paraffin-embedded tumor tissue surgically removed or obtained at autopsy. Twenty-one out of 22 carcinomas of the exocrine pancreas contained c-K-ras genes with mutations at codon 12. In seven cases tested, the mutation was present in both primary tumors and their corresponding metastases. No mutations were detected in normal tissue from the same cancer patients or in five gall bladder carcinomas. We conclude from these results that c-K-ras somatic mutational activation is a critical event in the oncogenesis of most, if not all, human cancers of the exocrine pancreas.
Adenocarcinoma↗
Analysis of genetic variability and mapping of point mutations in influenza virus by the RNase A mismatch cleavage method.
We have applied the RNase A mismatch cleavage method to analyze genetic variability in RNA viruses by using influenza virus as a model system. Uniformly labeled RNA probes synthesized from a cloned hemagglutinin gene of a given viral strain were hybridized to RNA isolated from other strains of characterized or uncharacterized genetic composition. The RNA.RNA heteroduplexes containing a variable number of base mismatches were digested with RNase A, and the resistant products were analyzed by denaturing polyacrylamide gel electrophoresis. We show that many of these single base mismatches are cleaved by RNase A, generating unique and characteristic patterns of resistant RNA fragments specific for each of the different viral strains. Comparative analysis of the cleavage patterns allows a qualitative estimation of the genetic relatedness and evolution of field strains. We also show that cleavage by RNase A at single base mismatches can readily detect and localize point mutations present in monoclonal antibody-resistant variants. This method should have wide applications in the study of RNA viruses, not only for epidemiological analysis but also in some diagnostic problems, such as characterization of phenotypic mutants.
Animals↗
Characterization of the human c-K-ras gene promoter.
The promoter of the human c-K-ras gene has been characterized by deletion mutagenesis in concert with stable and transient expression gene transfer experiments. The transcription initiation sites were determined by S1 mapping and RNase A protection experiments. The c-K-ras promoter region is rich in G + C, lacks TATA and CCAAT boxes and contains sequence similarities with other house-keeping genes such as the dihydrofolate reductase (DHFR) and the epidermal growth factor (EGF) receptor genes. The promoter of the c-K-ras gene consists of multiple elements and initiation of transcription occurs at multiple sites. A 54 bp DNA fragment immediately upstream from the 5' end untranslated exon controls the position of many of the transcription initiation sites and direct sufficient transcription for transformation of NIH3T3 cells. However, these sequences can be replaced by other upstream sequences which are required for optimal gene expression. In addition, sequences overlapping with the 5' end untranslated exon and therefore downstream from the major transcription initiation sites are important (although not sufficient) for transcription because their deletion greatly impairs the promoter activity of the upstream elements.
Animals↗
Initial characterization of a potential transcriptional enhancer for the human c-K-ras gene.
The nucleotide sequence of the 5' end distal region of the human c-K-ras gene promoter was determined. This region, coincident with a variable DNAse I hypersensitive site in native chromatin, contains sequence similarities with known enhancers. A 400 bp MstII DNA fragment of this region stimulated in cis the correctly initiated transcription of the human beta-globin gene in transfected Hela cells. The stimulation of beta-globin transcription (5-6 fold) was dependent on the distance and orientation of the c-K-ras sequences and on the presence of the CCAAT and CACCC elements in the beta-globin promoter. Interaction of nuclear factors with these c-K-ras sequences was analysed by DNAase I footprinting assays using Hela nuclear extracts. A protein binding to these sequences was identified as nuclear factor 1 (NF-1) by DNAase I competition footprinting experiments. However, disruption of the c-K-ras NF-1 binding site by insertion mutagenesis had no effect on the transcriptional activity of the c-K-ras element.
Base Sequence↗
Expression of the Kirsten ras viral and human proteins in Escherichia coli.
The expression vectors pINIII-A and pINIII (lpp p5) were used to construct plasmids which direct the synthesis in Escherichia coli of the Kirsten ras viral (v-Ki-ras) and human cellular (c-Ki-ras) oncogene products as fusion proteins containing 9 and 10 extra amino acids, respectively, at their N termini. Authenticity of the bacterially produced proteins was determined by immunoprecipitation and immunoblot analyses with ras-specific monoclonal antibodies. After induction with isopropyl-beta-D-thiogalactopyranoside, the viral protein represented approximately 20% of the total cellular protein. The majority of the protein was found in the postsonication low-speed centrifugation pellet. The synthesized viral protein was active in GTP binding, as judged by autophosphorylation and photoaffinity labeling assays.
DNA Restriction Enzymes↗
Chromatin structure of the promoter region of the human c-K-ras gene.
The chromatin structure of the human c-K-ras gene has been investigated in various cultured normal and tumor human cells and in a rat cell line transformed with the human oncogene. The promoter region is hypersensitive to DNAse I, micrococcal nuclease, endogenous nucleases and to S1 nuclease in supercoiled plasmids. This hypersensitive region is present in the different cell types analyzed and both normal and mutant alleles exhibit similar general sensitivity to DNAse I digestion in the same tumor cells. However, the 5' more distal DNAse I hypersensitive site, which is coincident with a region of the gene containing sequence homologies with known enhancers, exhibits variable sensitivity which appears to be higher in the tumor than in the normal and in the human than in the rat cells which we have analyzed. These data suggest the presence of specific factors interacting with the promoter sequences and delimits the transcription unit of the c-K-ras locus.
Cell Line↗
Oncogene amplification during tumorigenesis of established rat fibroblasts reversibly transformed by activated human ras oncogenes.
Normal rat fibroblasts of the established cell line Rat 4 were cotransformed with activated human ras oncogenes and with a cloned chicken thymidine kinase (tk) gene. Linkage between tk and ras genes allowed the isolation of oncogene deletion revertants and of cell clones showing varying degrees of malignant phenotype. Southern and Northern experiments in concert with tumorigenicity assays show that the malignant transformation of these cells by mutant ras oncogenes is a gradual but reversible process that depends on the relative abundance of oncogene sequences and their corresponding transcripts. We also show that moderate amplification of a c-K-ras oncogene in these cells results in a clear increase in their tumorigenicity and that the mutant gene present in low copy numbers in cultured cells undergoes amplification in the corresponding in vivo induced tumors.
Animals↗
Expression of intact Ki-ras p21 protein in Escherichia coli.
We have constructed recombinant plasmids capable of expressing in Escherichia coli the intact ras p21 protein encoded by Kirsten murine sarcoma virus. The Ki-ras gene was inserted into an expression vector carrying the E. coli tryptophan promoter and E. coli lipoprotein transcriptional terminator. The resulting plasmids direct the synthesis of large quantities of p21 protein, which represented 20% of the total cellular protein. The Ki-ras p21 protein is immunoprecipitated with monoclonal antibody to p21, and exhibits guanine nucleotide binding activity and autophosphorylation activity. The purified Ki-ras p21 expressed in E. coli has shown to have intact N-terminal and C-terminal amino acid sequences predicted by the nucleotide sequences and migrate as -23K in SDS/polyacrylamide gels.
Amino Acid Sequence↗
A method to detect and characterize point mutations in transcribed genes: amplification and overexpression of the mutant c-Ki-ras allele in human tumor cells.
A significant percentage of human tumors contain activated ras oncogenes that have acquired oncogenic potential as a result of somatic point mutations at codon 12 or 61 of the encoded ras gene product. We report here a method to detect and characterize mutations in ras genes that is based on the ability of pancreatic ribonuclease (RNase A; EC 3.1.27.5) to cleave RNA heteroduplexes containing single-base mismatches. Using this method, we show that certain human tumor cells contain mutant c-Ki-ras genes, and we define the nature and position of these mutations. At the same time, we describe the presence and estimate the expression of both normal and mutant c-Ki-ras alleles in the same tumor cells. This method should be useful for the diagnostic detection and characterization of single point mutations in expressed genes.
Alleles↗